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Subjects

Abstract

Fibro-adipogenic progenitors (FAPs) are typically activated in response to muscle injury, and establish functional interactions with inflammatory and muscle stem cells (MuSCs) to promote muscle repair. We found that denervation causes progressive accumulation of FAPs, without concomitant infiltration of macrophages and MuSC-mediated regeneration. Denervation-activated FAPs exhibited persistent STAT3 activation and secreted elevated levels of IL-6, which promoted muscle atrophy and fibrosis. FAPs with aberrant activation of STAT3–IL-6 signalling were also found in mouse models of spinal cord injury, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS) and in muscles of ALS patients. Inactivation of STAT3–IL-6 signalling in FAPs effectively countered muscle atrophy and fibrosis in mouse models of acute denervation and ALS (SODG93A mice). Activation of pathogenic FAPs following loss of integrity of neuromuscular junctions further illustrates the functional versatility of FAPs in response to homeostatic perturbations and suggests their potential contribution to the pathogenesis of neuromuscular diseases.

Acknowledgements

This work was supported by the Italian Ministry of Health (grant no. GR-2013-02356592) to L.M., NIH grants R01AR056712, R01AR052779, P30 AR061303, an MDA grant and EPIGEN grant to P.L.P., NIH grants R01 AR064873 and P30 AR061303 and MDA grant to A.S., California Institute for Regenerative Medicine (CIRM) training grant TG2-01162 and AFM-Téléthon Postdoctoral fellowship (no. 21084) to D.S. The authors thank E. Aleo at the Institute of Applied Genomics in Udine, Italy, for RNA–seq library preparation and sequencing, L. Battistini and G. Borsellino for flow cytometry related discussions and advice, L. Berghella for preliminary analysis, and R. Rizzi for support in in vivo treatments. The authors also thank D. Guttridge, S. Akira and S. Schenk for sharing mice generated in their laboratories, and C. Heil for help with graphic formatting. D.S. and U.E. contributed equally to this work.

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Contributions

Project conception and design was carried out by L.M. and P.L.P., denervation procedures and related experiments in vivo and ex vivo, data generation and analysis by L.M., M.P., F.L., D.P. and M.V.A., RNA–seq data analysis and statistics by C.N. and S.G., experiments with STAT3 fl/fl mice, ex vivo STAT3 deletion/blockade and related transplantation assays by D.S., U.E., L.M. and A.S., flow cytometry by L.M. and M.d.B., human ALS sample sourcing by R.R.-G., CD90 expression analysis in mouse and human FAPs by L.G. and L.M., experiments with SODG93A mice and ALS sample analysis by L.M. and M.P., experiments with SCI by S.M. and L.M., and experiments with SMA by V.P., C.S. and L.M. The manuscript was written by P.L.P., and funding was sourced by P.L.P. and L.M.

(a) MA-plots showing the fold change and normalized counts for FAPs from denervated (DEN 7 and 15 days) and injured (CTX) muscles, compared to control mice. Data show results from the mean of 2 animals/group (b) Top enriched pathways identified with IPA software, for differentially expressed genes unique to injury (CTX; left), denervation day 15 (DEN15d; right) or common to the two conditions (down). For each plot, x-axis represents the –log of the P-value (right-tailed Fisher’s exact test) and the orange dots on each pathway bar correspond to the ratio of the number of genes in a given pathway that meets the cutoff criteria, divided by the total number of genes belonging to that pathway.

(a) MA-plots showing the fold change and normalized counts for myofibers from 7 days denervated (DEN) muscles compared to control myofibers. Data show results from the mean of 2 animals/group (b) Expression heatmap of genes differentially expressed in myofibers derived from denervated (DEN) muscle compared to myofibers derived from Control (CTR) mice. Gene expression is represented as z-score calculated across the rows. Data show results from the mean of 2 animals/group (c) Upstream Regulators predicted to be differentially activated or inhibited in myofibers from denervated (DEN) myofibers compared to control (CTR) myofibers, by IPA analysis. (d) Top Canonical Pathways altered in myofibers from 7 days Denervated (DEN) muscles, compared to control myofibers. For each plot, x-axis represents the –log of the P-value (right-tailed Fisher’s exact test) and the orange dots on each pathway bar correspond to the ratio of the number of genes in a given pathway that meet the cutoff criteria, divided by the total number of genes belonging to that pathway. (e) Top Metabolic Pathways altered in Myofibers from 7 days Denervated (DEN) muscle compared to control myofibers (see Supp.Fig.3b for further details). For each plot, x-axis represents the –log of the P-value (right-tailed Fisher’s exact test) and the orange dots on each pathway bar correspond to the ratio of the number of genes in a given pathway that meets the cutoff criteria, divided by the total number of genes belonging to that pathway.